The central hypothesis of this proposal is that, in the Tumor Necrosis Factor-alpha (TNF)-activated pulmonary endothelium, reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO-) modulate the prolonged (greater than or equal to 4 hrs.) activation of Protein Kinase C-alpha (PKC-alpha), at least in part, by stimulating activator protein-1 (AP-1)-mediated transcription of PKCalpha. The pathogenetic sequence begins with RNS (either ONOO-, NO or both) activating guanylate cyclase, resulting in the generation of cGMP. The stimulated cGMP-PKG pathway phosphorylates the AP-1 subcomponents cFos and/or cJun, leading to AP-1 activation. The activated AP- 1 induces transcription of the PKCalpha gene. The increased transcription results in increased translation of PKCalpha mRNA into C Kinase protein, promoting the prolonged activation of PKC, with subsequent well-studied injurious effects on the pulmonary endothelium. The specific aims of the current proposal are: (1) to study whether TNF induces transcription of the pulmonary endothelial PKCalpha gene, (2) to determine the roles of RNS generation and AP-1 activation in the TNF induction of message for pulmonary endothelial PKCalpha, (3) to investigate whether the cGMP-PKG-mediated phosphorylation induced by TNF and RNS activates AP-1, and (4) to probe the roles of RNS, AP-1 and PKCalpha message in the prolonged activation of pulmonary endothelial PKCalpha induced by TNF. The hypothesis will be tested using human and bovine pulmonary microvessel endothelial cells. TNF-induced PKC-alpha transcription is studied using Nuclear Run-On and Northern Blot of cytosolic RNA. AP-1 activation is investigated using the Electrophoretic Mobility Shift Assay. Transfection with AP-1 reporter plasmids is used to verify role of RNS in TNF-induced AP-1 activation in gene regulation. Antisense oligonucleotides (anti-cJun, anti-PKC-alpha) is used to verify the AP-1-mediated maintenance of TNF-induced PKC-alpha transcription and activity. The role of RNS and PKG is investigated using (1) direct assay and (2) agonists and antagonists of RNS and PKG. Synthesis of PKC-alpha is studied using immunoprecipitation and Western Blot of 35S-labeled PKC, and activity of PKC is assessed by PKC translocation and phosphorylation of PKC-alpha specific substrate.